Analytical studies of 'mixed sequence' oligodeoxyribonucleotides synthesized by competitive coupling of either methyl- or beta-cyanoethyl-N,N-diisopropylamino phosphoramidite reagents, including 2'-deoxyinosine.

High-performance liquid chromatography (HPLC) and 1H/31P nuclear magnetic resonance (NMR) spectroscopy were used to measure the molar ratio of oligodeoxyribonucleotide products in mixtures obtained with automated DNA synthesizers that employed competitive coupling of either standard methyl- or newer beta-cyanoethyl-N,N-diisopropylamino phosphoramidite reagents, which include deoxyinosine. Mixtures of these reagents when used as freshly prepared solutions afforded ratios of products that indicated negligibly small differences among the rates of the various competitive coupling reactions. However, studies of reagent stability in solution revealed that both types of the N-isobutyryl deoxyguanosine reagent decompose faster than their corresponding dA, dC, and dT phosphoramidites, which led to significantly lower proportions of dG-containing sequences. This problem was attenuated for the beta-cyanoethyl reagents due to their slower rate of decomposition.

Nuclear magnetic resonance and circular dichroism studies of a duplex--single-stranded hairpin loop equilibrium for the oligodeoxyribonucleotide sequence d(CGCGATTCGCG).

Nuclear magnetic resonance (NMR) and circular dichroism (CD) studies have been carried out with the oligodeoxyribonucleotide mismatch sequence, d(CGCGATTCGCG), 1. It has been found that 1 exists, in solution, as an equilibrium mixture of slowly interconverting, structured conformational isomers, 1a and 1b. On the basis of the concentration dependence of the 1a-1b equilibrium, the 1H NMR spectrum of the imino protons of the nucleotide bases, and the individual CD spectra of 1a and 1b, it is suggested that the two species correspond to a B-type DNA duplex and a single-stranded, hairpin-loop structure; the portion of the single-stranded species not involved in the loop appears to have a B-type DNA structure (on the basis of the CD measurements). To facilitate 1H NMR resonance assignments, the two possible des-methyl thymidine derivatives of 1 were synthesized; the effect of this substitution on the physical chemical properties of 1 was explored. The 1H NMR spectra of 1, as a function of temperature, showed that, under conditions wherein both species were present to a significant extent, the duplex form melted at a lower temperature than the single-stranded, hairpin loop structure.